Evidence for Cytochrome P4502e1 As A Source Of Catalytic Iron In Oxidant-Induced Nephrotoxicity ﻣﺠﻤﻮﻋﺔ إن ﻋﻠﻰ اﻟﺪﻻﻟﺔ ٢ اي ١ ب اﻟﺴﺎﯾﺘﻮﻛﺮوم ﻻﻧﺰﯾﻢ ٤٥٠ اﻟﻌﺎﻣﻞ ﻟﻠﺤﺪﯾﺪ ﻛﻤﺼﺪر ﺗﻌﻤﻞ اﻟﻤﺆﻛﺴﺪة ﺑﺎﻟﻤﻮاد اﻟﻜﻠﻮي اﻟﺘﺴﻤﻢ ﻓﻲ اﻟﻤﺴﺎﻋﺪ (original) (raw)
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CYP3A induction aggravates endotoxemic liver injury via reactive oxygen species in male rats
Free Radical Biology and Medicine, 2004
We carried out this experiment to evaluate the relationship between isoforms of cytochrome P450 (P450) and liver injury in lipopolysaccharide (LPS)-induced endotoxemic rats. Male rats were intraperitoneally administered phenobarbital (PB), a P450 inducer, for 3 days, and 1 day later, they were intravenously given LPS. PB significantly increased P450 levels (200% of control levels) and the activities (300 -400% of control) of the specific isoforms (CYP), CYP3A2 and CYP2B1, in male rats. Plasma AST and ALT increased slightly more in PB-treated rats than in PBnontreated (control) rats with LPS treatment. Furthermore, either troleandomycin or ketoconazole, specific CYP3A inhibitors, significantly inhibited LPS-induced liver injury in control and PB-treated male rats. To evaluate the oxidative stress in LPS-treated rats, in situ superoxide radical detection using dihydroethidium (DHE), hydroxy-2-nonenal (HNE)modified proteins in liver microsomes and 8-hydroxydeoxyguanosine (8-OHdG) in liver nuclei were measured in control and PB-treated rats. DHE signal intensity, levels of HNE-modified proteins, and 8-OHdG increased significantly in PBtreated rats. LPS further increased DHE intensity, HNE-modified proteins, and 8-OHdG levels in normal and PB-treated groups. CYP3A inhibitors also inhibited the increases in these items. Our results indicate that the induction or preservation of CYP isoforms further promotes LPS-induced liver injury through mechanisms related to oxidative stress. In particular, CYP3A2 of P450 isoforms made an important contribution to this LPS-induced liver injury. D 2004 Elsevier Inc. All rights reserved.
Toxicology in Vitro, 2003
Deliberate exposure to solvents has been associated with kidney disorders. However, the mechanism by which solvents induce renal damage after acute exposure has not been studied. Proximal tubular cell (LLC-PK1) cytotoxicity after exposure for 48 h to either 5 mm of p-xylene (XY) or toluene (TL) was compared to control (C) by cell viability (MTS assay), LDH release, DNA fragmentation, and malondialdehyde (MDA) release. CYP2E1 activity with or without a free radical scavenger (catalase-CT), or the CYP2E1 inhibitor disulfiram (DSF), was examined. Both p-xylene and toluene significantly reduced cell viability (XY 53.9 8 AE1.6 vs TL 54.8AE 0.9 vs C 102.7 AE 2.1), increased CYP2E1 activity (mm/mg protein/min) (XY 3.6 AE 0.5 vs TL 3.7 AE0.7 vs C 1.3 AE0.4) and MDA release (mm/mg protein) (XY 29.1 AE3.9 vs TL 12.3AE 1.4 vs C 2.8 AE 0.3). LDH was increased (XY 59.9 AE 3.0 vs TL 27.6 AE 0.5 vs C 8.4 AE 1.2), but there was no significant change in DNA fragmentation (OD/mg protein) suggesting necrosis as the predominant mode of cell death. DSF significantly attenuated CYP2E1 activity (XY+DSF 1.4 AE0.9, TL+DSF 2.3AE 0.1), LDH release (XY+DSF 45.1 AE2.0, TL+DSF 13.0 AE 0.2) and MDA release (XY+DSF 4.3AE 0.5, TL+CT 6.2 AE 1.1). Moreover, CT attenuated LDH release (XY+CT 36.4AE 5.1, TL+DSF 15.6 AE0.5) and MDA release (XY+DSF 5.4 AE0.7, TL+DSF 6.6 AE 1.3) in XY and TL treated cells. This study confirms the pivotal role of CYP2E1 in solvent-induced oxidative stress and necrosis in proximal tubular cells after exposure to solvent at 5 mm for 48 h. #
Molecular Medicine Reports, 2020
During the reperfusion phase of ischemia-reperfusion injury, reactive oxygen species (ROS) production aggravates the course of many diseases, including acute kidney injury. Among the various enzymes implicated in ROS production are the enzymes of the cytochromes P450 superfamily (CYPs). Since arylhydrocarbon receptor (AhR) controls the expression of certain CYPs, the involvement of this pathway was evaluated in reperfusion injury. Because AhR may interact with the nuclear factor erythroid 2-related factor 2 (Nrf2) and the hypoxia-inducible factor-1α (HIF-1α), whether such an interaction takes place and affects reperfusion injury was also assessed. Proximal renal proximal tubular epithelial cells were subjected to anoxia and subsequent reoxygenation. At the onset of reoxygenation, the AhR inhibitor CH223191, the HIF-1α activator roxadustat, or the ferroptosis inhibitor α-tocopherol were used. The activity of AhR, Nrf2, HIF-1α, and their transcriptional targets were assessed with western blotting. ROS production, lipid peroxidation and cell death were measured with colorimetric assays or cell imaging. Reoxygenation induced ROS production, lipid peroxidation and cell ferroptosis, whereas CH223191 prevented all. Roxadustat did not affect the above parameters. Reoxygenation activated AhR and increased CYP1A1, while CH223191 prevented both. Reoxygenation with or without CH223191 did not alter Nrf2 or HIF-1α activity. Thus, AhR is activated during reoxygenation and induces ROS production, lipid peroxidation and ferroptotic cell death. These detrimental effects may be mediated by AhR-induced CYP overexpression, while the Nrf2 or the HIF-1α pathways remain unaffected. Accordingly, the AhR pathway may represent a promising therapeutic target for the prevention of reperfusion injury.
Current Pharmaceutical Design, 2006
Ischemia-reperfusion (IR) injury is a multifactorial process triggered when the liver or other organs are transiently subjected to reduced blood supply followed by reperfusion. It has been shown that "reactive oxygen species" (ROS) are generated during ischemia and reperfusion and may represent pivotal mediators of the ensuing pathological complications. In some cases, however, moderate production of ROS may exert protective effects, a phenomenon presumably related to "ischemic preconditioning". This review will focus mainly on: a) describing the sources and the biochemical mechanisms of ROS generation during ischemia and reperfusion, b) discussing current developments in understanding the biochemical pathways by which ROS may induce toxic or protective effects, c) critically evaluating the results of previous attempts to counteract the toxic effects of ROS by using a variety of antioxidant and transition metalchelating agents, and d) if feasible, proposing potential new pharmaceutical agents aimed at ameliorating ROS-inducing deleterious effects during reperfusion. It is concluded that ROS are generated from different sources, at different periods during IR, and may act by a variety of not well understood biochemical mechanisms which ultimately lead to cell damage and tissue failure.
Role of Calcium and Calcium-activated Proteases in CYP2E1-dependent Toxicity in HEPG2 Cells
Journal of Biological Chemistry, 2001
The objective of this work was to investigate whether CYP2E1-and oxidative stress-dependent toxicity in HepG2 cells is mediated by an increase of cytosolic Ca 2؉ and activation of Ca 2؉-modulated processes. HepG2 cells expressing CYP2E1 (E47 cells) or control cells not expressing CYP2E1 (C34 cells) were preloaded with arachidonic acid (AA, up to 10 M) and, after washing, incubated with iron-nitrilotriacetic acid (up to 100 M) for variable periods (up to 12 h). Toxicity was greater in E47 cells than in C34 cells at all times and combinations of iron/AA tested. Cytosolic calcium increased with incubation time in both cell lines, but the increase was higher in E47 cells than in C34 cells. The rise in calcium was an early event and preceded the developing toxicity. Toxicity in E47 cells and the increase in Ca 2؉ were inhibited by omission of Ca 2؉ from the extracellular medium, and toxicity was restored by reincorporation of Ca 2؉. An inhibitor of Ca 2؉ release from intracellular stores did not prevent the toxicity or the increase in Ca 2؉ , reflecting a role for the influx of extracellular Ca 2؉ in the toxicity. Reactive oxygen production was similar in media with or without calcium, indicating that calcium was not modulating CYP2E1-dependent oxidative stress. Toxicity, lipid peroxidation, and the increase of Ca 2؉ in E47 cells exposed to iron-AA were inhibited by ␣-tocopherol. E47 cells (but not C34 cells) exposed to iron-AA showed increased calpain activity in situ (40fold). The toxicity in E47 cells mirrorred calpain activation and was inhibited by calpeptin, suggesting that calpain activation plays a causal role in toxicity. These results suggest that CYP2E1-dependent toxicity in this model depends on the activation of lipid peroxidation, followed by an increased influx of extracellular Ca 2؉ and activation of Ca 2؉-dependent proteases.
Hydrogen peroxide is an important regulator of protein tyrosine phosphatase activity via reversible oxidation. However, the role of iron in this reaction has not been yet elucidated. Here we compare the influence of hydrogen peroxide and the ferrous iron (reagent for Fenton reaction) on the enzymatic activity of recombinant CD45, LAR, PTP1B phosphatases and cellular CD45 in Jurkat cells. The obtained results show that ferrous iron (II) is potent inhibitor of CD45, LAR and PTP1B, but the inhibitory effect is concentration dependent. We found that the higher concentrations of ferrous iron (II) increase the inactivation of CD45, LAR and PTP1B phosphatase caused by hydrogen peroxide, but the addition of the physiological concentration (500 nM) of ferrous iron (II) has even a slightly preventive effect on the phosphatase activity against hydrogen peroxide.
Role of cytochrome P-450 in reperfusion injury of the rabbit lung
Journal of Clinical Investigation, 1990
Reactive oxygen species are a major cause of damage occurring in ischemic tissue after reperfusion. During reperfusion transitional metals such as iron are required for reactive oxygen species to mediate their major toxic effects. Xanthine oxidase is an important source of reactive oxygen species during ischemia-reperfusion injury, but not in all organs or species. Because cytochrome P450 enzymes are an important pulmonary source of superoxide anion (02) generation under basal conditions and during hyperoxia, and provide iron catalysts necessary for hydroxyl radical COH) formation and propagation of lipid peroxidation, we postulated that cytochrome P450 might have a potential role in mediating ischemia-reperfusion injury. In this report, we explored the role of cytochrome P450 enzymes in a rabbit model of reperfusion lung injury. The P450 inhibitors 8-methoxypsoralen, piperonyl butoxide, and cimetidine markedly decreased lung edema from transvascular fluid flux. Cimetidine prevented the reperfusion-related increase in lung microvascular permeability, as measured by movement of '25I-albumin from the vascular space into lung water and alveolar fluid. P450 inhibitors also prevented the increase in lung tissue levels of thiobarbituric acid reactive products in the model. P450 inhibitors did not block enhanced 0°generation by ischemic reperfused lungs, measured by in vivo reduction of succinylated ferricytochrome c in lung perfusate, but did prevent the increase in non-protein-bound low molecular weight chelates of iron after reperfusion. Thus, cytochrome P450 enzymes are not likely a major source of enhanced 05 generation, but serve as an important source of iron in mediating oxidant injury to the rabbit lung during reperfusion. These results suggest an important role of cytochrome P450 in reperfusion injury to the lung and suggest potential new therapies for the disorder.
Extracellular Iron(II) Can Protect Cells from Hydrogen Peroxide
Archives of Biochemistry and Biophysics, 1996
We hypothesized that exposure of cells to H 2 O 2 plus It is well known that H 2 O 2 plus Fe 2/ will generate Fe 2/ would increase formation of cell-derived lipid peroxoxidants that initiate free radical reactions and that ides that would inactivate prostaglandin H synthase, rethese radicals may result in cell injury (1, 2). The prisulting in decreased prostaglandin synthesis. Therefore, mary oxidant generated by H 2 O 2 plus Fe 2/ is the hywe treated human endothelial cells with 0-100 mM H 2 O 2 droxyl radical (HO •) (3). followed immediately by addition of 0-200 mM Fe 2/. After oxidant exposure, cells were stimulated with 20 mM ara-Fe 2/ / H 2 O 2 r Fe 3/ / OH 0 / HO • (Fenton reaction) chidonic acid to induce prostaglandin I 2 (PGI 2) synthesis. The hydroxyl radical is highly reactive and is believed Adding 100 mM H 2 O 2 prior to arachidonic acid decreased to cause cytolysis by initiating peroxidation of cell lip-PGI 2 synthesis more than 80%. However, to our surprise, ids (4-6). the addition of Fe 2/ , in increasing amounts, progressively protected PGI 2 synthesis against the harmful effects of In addition to HO • , the reaction of H 2 O 2 with Fe 2/ H 2 O 2. A ratio of one part H 2 O 2 to two parts Fe 2/ offered will generate Fe 2/ /Fe 3/ ratios that may increase lipid almost complete protection, whereas Fe 3/ did not protect oxidation (7, 8). A 1:1 ratio of Fe 2/ to Fe 3/ has been PGI 2 synthesis from H 2 O 2. We found that 100 mM H 2 O 2 reported as ideal for maximizing lipid peroxidation (7was not cytolytic; however, 250 mM H 2 O 2 was cytolytic; 9). If a reaction mixture contains two parts Fe 2/ to one Fe 2/ protected against this cytotoxicity. In addition, expart H 2 O 2 , the products would be HO • , plus a 1:1 ratio tracellular Fe 2/ prevented the rise in intracellular calof Fe 2/ :Fe 3/. The resulting combination of HO • plus a cium caused by H 2 O 2 and extracellular Fe 2/ preserved 1:1 ratio of Fe 2/ :Fe 3/ might generate more lipid peroxintracellular glutathione in H 2 O 2-exposed cells. Electron ides than HO • alone. In addition, the Fe 2/ :Fe 3/ ratios paramagnetic resonance spin trapping demonstrated may generate the perferryl ion, Fe 2/ OO • S Fe 3/ O r0 2 , that extracellular Fe 2/ generated the hydroxyl free radiwhich may generate additional lipid peroxides (2, 10, cal, HO • , outside the cell. We speculate that extracellular 11). Therefore, it seems likely that a ratio of two parts Fe 2/ protects the intracellular space from H 2 O 2 by initiat-Fe 2/ to one part H 2 O 2 would increase cytolysis over ing the Fenton reaction outside the cell. This reductive either agent alone. cleavage of H 2 O 2 generates HO • in the extracellular Lipid peroxidation products may also injure specific space, where much of the HO • will react with noncellular cell functions without causing cytolysis. For example, components, thereby protecting the cell interior. ᭧ 1996 prostaglandin H synthase (PGHS) 2 is an enzyme that Academic Press, Inc.
The role of cytochrome P450 (CYP) enzymes in hyperoxic lung injury
Expert Opinion on Drug Metabolism & Toxicology, 2020
Introduction: Hyperoxic lung injury is a condition that can occur in patients in need of supplemental oxygen, such as premature infants with bronchopulmonary dysplasia or adults with acute respiratory distress syndrome. Cytochrome P450 (CYP) enzymes play critical roles in the metabolism of endogenous and exogenous compounds. Areas covered: Through their complex pathways, some subfamilies of these enzymes may contribute to or protect against hyperoxic lung injury. Oxidative stress from reactive oxygen species (ROS) production is most likely a major contributor of hyperoxic lung injury. CYP1A enzymes have been shown to protect against hyperoxic lung injury while CYP1B enzymes seem to contribute to it. CYP2J2 enzymes help protect against hyperoxic lung injury by triggering EET production, thereby, increasing antioxidant enzymes. The metabolism of arachidonic acid to ωterminal hydroxyeicosatetraenoic acid (20-HETEs) by CYP4A and CYP4F enzymes could impact hyperoxic lung injury via the vasodilating effects of 20-HETE. CYP2E1 and CYP2A enzymes may contribute to the oxidative stress in the lungs caused by ethanol-and nicotine-metabolism, respectively. Expert opinion: Overall, the CYP enzymes, depending upon the isoform, play a contributory or protective role in hyperoxic lung injury, and are, therefore, ideal candidates for developing drugs that can treat oxygen-mediated lung injury.
Free Radical Biology and Medicine, 2010
Heme oxygenase (HO-1), the rate-limiting enzyme in the physiological breakdown of heme, is ubiquitous, and its expression can be increased by arsenite [As(III)], and similar other stimuli that induce cellular oxidative stress. Interestingly, it has been shown that the As(III)-induced HO-1 is inversely correlated with a decrease in cytochromes P450 (P450s) activity; however, the direct role for HO-1 in the inhibition of P450 enzymes remains unknown. Our results showed that As(III) at a concentration of 5 M decreased the constitutive and inducible expression of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 at the mRNA, protein, and catalytic activity levels. Moreover, As(III) decreased the nuclear accumulation of aryl hydrocarbon receptor (AhR) and pregnane X receptor without increasing their degradation. As(III) also increased the binding of cytosolic AhR to heat shock protein 90 and hepatitis B virus X-associated protein 2. In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin as an inducer for CYP1A and rifampin as an inducer for CYP3A, As(III) decreased the enzymatic activity of the four P450s more than it decreased their mRNA or protein expression levels. It is noteworthy that treatment with the competitive HO-1 inhibitor, tin-mesoporphyrin, or supplementing external heme partially reversed the As(III)-mediated decrease in activities of the four P450s. In conclusion, the current study provides the first evidence that As(III) decreases CYP1A1, CYP1A2, CYP3A23, and CYP3A2 expression in freshly isolated rat primary hepatocytes. Furthermore, inhibiting the As(III)-mediated induction of HO-1 partially restores the enzymatic activity of these P450s that was initially decreased by As(III), confirming the direct role of HO-1 in the inhibition of P450s.